Abstract

Preferentially expressed antigen in melanoma (PRAME) is a cancer-testis antigen that is expressed in many cancers and leukemias. In healthy tissue, PRAME expression is limited to the testes and ovaries, making it a highly attractive cancer target. PRAME is an intracellular protein that cannot currently be drugged. After proteasomal processing, the PRAME300–309 peptide ALYVDSLFFL (ALY) is presented in the context of human leukocyte antigen HLA-A*02:01 molecules for recognition by the T cell receptor (TCR) of cytotoxic T cells. Here, we have described Pr20, a TCR mimic (TCRm) human IgG1 antibody that recognizes the cell-surface ALY peptide/HLA-A2 complex. Pr20 is an immunological tool and potential therapeutic agent. Pr20 bound to PRAME+HLA-A2+ cancers. An afucosylated Fc form (Pr20M) directed antibody-dependent cellular cytotoxicity against PRAME+HLA-A2+ leukemia cells and was therapeutically effective against mouse xenograft models of human leukemia. In some tumors, Pr20 binding markedly increased upon IFN-γ treatment, mediated by induction of the immunoproteasome catalytic subunit β5i. The immunoproteasome reduced internal destructive cleavages within the ALY epitope compared with the constitutive proteasome. The data provide rationale for developing TCRm antibodies as therapeutic agents for cancer, offer mechanistic insight on proteasomal regulation of tumor-associated peptide/HLA antigen complexes, and yield possible therapeutic solutions to target antigens with ultra-low surface presentation.

Figure 1

Pr20 binds ALY/HLA-A2 complexes and PRAME+HLA-A2+ leukemia.

Pr20 was directly labeled by conjugation to the fluorophore APC. (A) TAP-deficient T2 cells were pulsed overnight with 50 μg/ml of ALY peptide or irrelevant control EW peptide or left unpulsed. Flow cytometry was used to determine P20 binding. (B) Each nonanchor residue in the ALY peptide was substituted for alanine, and peptides were pulsed onto T2. Pr20 binding was determined by flow cytometry relative to native ALY peptide–pulsed T2. Cell-surface HLA-A2 was also measured by flow cytometry to ensure altered peptides maintained the ability to bind and stabilize HLA-A2 compared with unpulsed T2. (C) PRAME mRNA expression was determined by qPCR, and samples that did not amplify after 40 cycles were considered negative. (D) The indicated cell lines were stained with Pr20 or an isotype control Ab, and binding was determined by flow cytometry. Surface HLA-A2 was also assessed compared with an isotype control. All data from A–D are representative of at least 3 experiments. (E) Whole blood populations from HLA-A2+ healthy donors were stained with Pr20 to determine possible crossreactivity. A representative gating strategy and Pr20 histogram compared with isotype control are shown, and data from all HLA-A2+ healthy donors (n = 5) are summarized. Staining was performed once independently for each healthy donor and an AML14 PRAME+HLA-A2+ leukemia–positive control was included in each assay to ensure assay reliability. SSC, side scatter; FSC, forward scatter.